PROJECT SUMMARY Approximately 40 million people are currently infected by HIV, with an additional 1.7 million people newly infected each year. While only a single person has been functionally cured of HIV, advances in antiretroviral therapy (ART) have drastically decreased AIDS-related illnesses and deaths for individuals on ART. However, HIV+ patients on ART still face debilitating AIDS-independent diseases, including HIV-associated neurocognitive disorders (HAND). HAND refers to a spectrum of three neurocognitive disorders that influence survival, quality of life, and everyday function: asymptomatic neurocognitive impairment (ANI), mild neurocognitive disorder (MND), and HIV-associated dementia (HAD). While the number of patients with the more severe forms of HAND have declined since the introduction of ART, an estimated 15-55% of HIV+ patients taking ART still develop a neurocognitive disorder. Patients with ANI are two to six times more likely to progress to a more severe form of HAND when compared to HIV+ patients who are neurocognitively normal. Thus, HAND remains an important and prevalent HIV-associated disease to affect individuals in the ART era. It is now thought that HAND develops because of functional changes in neurons caused by chronic inflammation and HIV infection of an immune cell in the brain, microglia. Though they are not a neuronal cell, microglia do play an important role in the general health and function of neural tissue. The immune responses of microglia are thought to be tightly regulated and controlled as to not normally harm the surrounding neurons. We do know that HIV can infect microglia. However, little is known about the progression of acute and chronic HIV infection in microglia nor how and what impact these infected cells have on surrounding neuronal tissue. Moreover, HIV infection is frequently associated with intravenous drug us such as heroin along with the growing abuse of prescription opioids. It remains unclear how the use of these drugs alters both neuronal and innate immune signaling and further contributes to HAND. One major roadblock in understanding infection of microglia has been the lack of systems for analysis in culture, as well as means for studying their impact on human brain functions. Recent developments in human pluripotent stem cell (hPSC)-derived microglia and 3D-brain organoids have opened new doors to understand HIV infection in these otherwise intractable cells. We have begun to bridge this knowledge gap by leveraging our strengths in HIV and brain organoid biology to model HAND in culture, where we can finally begin to answer important questions in the roles of HIV, opioids, and microglia and other cells in this debilitating HIV- associated disease. Success of our proposed research will 1) define the response of microglia to HIV infection and opioid treatment, 2) characterize how dysregulated microglia affect brain organoid structure, neural network health, and signaling, and 3) establish a foundation for therapeutic discovery to reduce neuroinflammation and HAND.